Lifelong impact of ENPP1 Deficiency and the early onset form of ABCC6 Deficiency from patient or caregiver perspective.

The ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) and ATP-binding cassette subfamily C member 6 (ABCC6) proteins play a prominent role in inhibiting ectopic calcification and arterial stenosis. Patients with ENPP1 Deficiency or infant onset ABCC6 Deficiency often present with pathological calcification, narrowed blood vessels, multiorgan dysfunction and high infant mortality. The heterogenous presentation and progression is well documented. Our objective was to characterize how these morbidities lead to burden of illness and poor quality of life across ages from the patient/caregiver perspective. Patients/caregivers were interviewed via phone using Institutional Review Board-approved questionnaires. Patient-reported outcomes were collected via validated instruments. Thirty-one caregivers and 7 patients participated: infant onset ABCC6 Deficiency, n = 6 (infants/children); ENPP1 Deficiency, n = 32 (13 infants, 12 children, 7 adults). ENPP1 and ABCC6-deficient children aged <8 years and aged 8-18 years reported poor school functioning (0.69 vs 0.72 effect size, respectively) and poor physical health (0.88 vs 1, respectively). In the total ENPP1 cohort, 72% (23/32) reported bone/joint pain and/or mobility/fatigue issues. Three of seven ENPP1-deficient adults reported moderate to severe pain (>4), as measured by the Brief Pain Inventory (BPI), that interfered with daily activities despite pain medication. Top reported burdens for caregivers of infants with ABCC6/ENPP1 Deficiencies included heart-related issues and hospitalizations. Treatment/medications, and hearing loss were the highest burdens reported by caregivers/families of the pediatric ENPP1 Deficiency cohort, whereas adults reported bone/joint pain and mobility impairment as the greatest burdens. Individuals with ENPP1 Deficiency or infant onset ABCC6 Deficiency experience lifelong morbidity causing substantial physical and emotional burden to patients/caregivers.

Ectopic Calcification and Hypophosphatemic Rickets: Natural History of ENPP1 and ABCC6 Deficiencies.

Generalized arterial calcification of infancy (GACI) is a rare disorder caused by ENPP1 or ABCC6 variants. GACI is characterized by low pyrophosphate, arterial calcification, and high mortality during the first year of life, but the natural course and possible differences between the causative genes remain unknown. In all, 247 individual records for patients with GACI (from birth to 58.3 years of age) across 19 countries were reviewed. Overall mortality was 54.7% (13.4% in utero or stillborn), with a 50.4% probability of death before the age of 6 months (critical period). Contrary to previous publications, we found that bisphosphonate treatment had no survival benefit based on a start-time matched analysis and inconclusive results when initiated within 2 weeks of birth. Despite a similar prevalence of GACI phenotypes between ENPP1 and ABCC6 deficiencies, including arterial calcification (77.2% and 89.5%, respectively), organ calcification (65.8% and 84.2%, respectively), and cardiovascular complications (58.4% and 78.9%, respectively), mortality was higher for ENPP1 versus ABCC6 variants (40.5% versus 10.5%, respectively; p = 0.0157). Higher prevalence of rickets was reported in 70.8% of surviving affected individuals with ENPP1 compared with that of ABCC6 (11.8%; p = 0.0001). Eleven affected individuals presenting with rickets and without a GACI diagnosis, termed autosomal recessive hypophosphatemic rickets type 2 (ARHR2), all had confirmed ENPP1 variants. Approximately 70% of these patients demonstrated evidence of ectopic calcification or complications similar to those seen in individuals with GACI, which shows that ARHR2 is not a distinct condition from GACI but represents part of the spectrum of ENPP1 deficiency. Overall, this study identified an early mortality risk in GACI patients despite attempts to treat with bisphosphonates, high prevalence of rickets almost exclusive to ENPP1 deficiency, and a spectrum of heterogenous calcification and multiple organ complications with both ENPP1 and ABCC6 variants, which suggests an overlapping pathology. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

The high expression of NUDT5 indicates poor prognosis of breast cancer by modulating AKT / Cyclin D signaling.

NUDIX hydrolase type 5 (NUDT5) is a kind of ADP-ribose pyrophosphatase and nucleotide metabolizing enzyme in cell metabolism. Previous studies have shown NUDT5 expression affected chromosome remodeling, involved in cell adhesion, cancer stem cell maintenance and epithelial to mesenchyme transition in breast cancer cells. Nevertheless, the role of NUDT5 in breast cancer progression and prognosis has not yet been systematically studied. This study explored the association of NUDT5 with the tumor development and poor prognosis in patients with breast cancer. Our results show that the levels of NUDT5 were upregulated in breast cancer cell lines and breast tumor tissues, and the expression of NUDT5 in breast tumor tissues increased significantly when compared with adjacent non-tumorous tissues by immunohistochemical staining of tissue microarrays. Breast cancer patients with high NUDT5 expression had a worse prognosis than those with low expression of NUDT5. In addition, the knockdown of NUDT5 suppressed breast cancer cell lines proliferation, migration and invasion, and dramatically inhibited the AKT phosphorylation at Thr308 and expression of Cyclin D1. The opposite effects were observed in vitro following NUDT5 rescue. Our findings indicated that the high expression of NUDT5 is probably involved in the poor prognosis of breast cancer via the activation of the AKT / Cyclin D pathways, which could be a prognostic factor and potential target in the diagnosis and treatment of breast cancer.

Osteoblast-specific deficiency of ectonucleotide pyrophosphatase or phosphodiesterase-1 engenders insulin resistance in high-fat diet fed mice.

Supraphysiological levels of the osteoblast-enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase-1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast-specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6-week-old mice lacking osteoblast NPP1 expression (osteoblast-specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast-specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed (p < .05). Male osteoblast-specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin-sensitizing under-carboxylated osteocalcin (195% increase; p < .05). However, following high-fat-diet challenge, osteoblast-specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity.

Encefalopatía epiléptica infantil precoz por mutación en ITPA / Early infantile epileptic encephalopathy due to ITPA mutation

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Cost-effectiveness analysis of pretreatment screening for NUDT15 defective alleles.

BACKGROUND: Nucleotide triphosphate diphosphatase (NUDT15) genetic testing in addition to thiopurine methyl transferase (TPMT) is recommended to reduce the incidence of adverse severe myelotoxicity episodes induced by thiopurines. OBJECTIVE: We assessed the cost-effectiveness ratio of combined screening for TMPT and NUDT15 defective alleles by genotyping or next-generation sequencing (NGS) using TPMT genotyping as the reference. Because of the genetic differences in thiopurine toxicity, we tested the screening strategies on individuals of Caucasian and Asian descent. METHODS: A decision tree compared conventional TPMT genotyping with combined TPMT/NUDT15 genotyping or NGS using a Monte-Carlo microsimulation model of patients with inflammatory bowel disease. The main outcome was the incremental cost-effectiveness ratios (ICER) with effectiveness being one averted severe myelotoxicity requiring hospitalization. RESULTS: The mean estimated cost of the TPMT genotyping for one year is twice in Asian compared with Caucasian patients (980 euro/patient versus 488 euro/patient), and the effectiveness of TPMT genotyping in Caucasian avoided 43 severe myelosuppressions per 10 000 patients over a year compared with 3.6 per 10 000 patients in Asian. Combined TPMT/NUDT15 genotyping compared with TPMT genotyping had an ICER of 7 491 281 euro per severe myelotoxicity averted in Caucasian, compared to 619 euro in Asian. The ICER of the NGS-based screening strategy is disproportionally high compared with genotyping, irrespective of ethnic descent. CONCLUSION: With a low cost-effectiveness threshold, combined screening for NUDT15 and TPMT defective alleles is cost-effective compared to TMPT screening alone in patients of Asian descent, but is unrealistic from a cost-effectiveness point of view in Caucasians.

ATP-based therapy prevents vascular calcification and extends longevity in a mouse model of Hutchinson-Gilford progeria syndrome.

Pyrophosphate deficiency may explain the excessive vascular calcification found in children with Hutchinson-Gilford progeria syndrome (HGPS) and in a mouse model of this disease. The present study found that hydrolysis products of ATP resulted in a <9% yield of pyrophosphate in wild-type blood and aortas, showing that eNTPD activity (ATP → phosphate) was greater than eNPP activity (ATP → pyrophosphate). Moreover, pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis (via TNAP; pyrophosphate → phosphate) was increased in both aortas and blood obtained from mice with HGPS. The reduced production of pyrophosphate, together with the reduction in plasma ATP, resulted in marked reduction of plasma pyrophosphate. The combination of TNAP inhibitor levamisole and eNTPD inhibitor ARL67156 increased the synthesis and reduced the degradation of pyrophosphate in aortas and blood ex vivo, suggesting that these combined inhibitors could represent a therapeutic approach for this devastating progeroid syndrome. Treatment with ATP prevented vascular calcification in HGPS mice but did not extend longevity. By contrast, combined treatment with ATP, levamisole, and ARL67156 prevented vascular calcification and extended longevity by 12% in HGPS mice. These findings suggest a therapeutic approach for children with HGPS.

Inositol pyrophosphates impact phosphate homeostasis via modulation of RNA 3' processing and transcription termination.

Fission yeast phosphate acquisition genes pho1, pho84, and tgp1 are repressed in phosphate-rich medium by transcription of upstream lncRNAs. Here, we show that phosphate homeostasis is subject to metabolite control by inositol pyrophosphates (IPPs), exerted through the 3'-processing/termination machinery and the Pol2 CTD code. Increasing IP8 (via Asp1 IPP pyrophosphatase mutation) de-represses the PHO regulon and leads to precocious termination of prt lncRNA synthesis. pho1 de-repression by IP8 depends on cleavage-polyadenylation factor (CPF) subunits, termination factor Rhn1, and the Thr4 letter of the CTD code. pho1 de-repression by mutation of the Ser7 CTD letter depends on IP8. Simultaneous inactivation of the Asp1 and Aps1 IPP pyrophosphatases is lethal, but this lethality is suppressed by mutations of CPF subunits Ppn1, Swd22, Ssu72, and Ctf1 and CTD mutation T4A. Failure to synthesize IP8 (via Asp1 IPP kinase mutation) results in pho1 hyper-repression. Synthetic lethality of asp1Δ with Ppn1, Swd22, and Ssu72 mutations argues that IP8 plays an important role in essential 3'-processing/termination events, albeit in a manner genetically redundant to CPF. Transcriptional profiling delineates an IPP-responsive regulon composed of genes overexpressed when IP8 levels are increased. Our results establish a novel role for IPPs in cell physiology.

Small alarmones (p)ppGpp regulate virulence associated traits and pathogenesis of Salmonella enterica serovar Typhi.

How Salmonella enterica serovar Typhi (S. Typhi), an important human pathogen, survives the stressful microenvironments inside the gastrointestinal tract and within macrophages remains poorly understood. We report here that S. Typhi has a bonafide stringent response (SR) system, which is mediated by (p)ppGpp and regulates multiple virulence-associated traits and the pathogenicity of the S. Typhi Ty2 strain. In an iron overload mouse model of S. Typhi infection, the (p)ppGpp0 (Ty2ΔRelAΔSpoT) strain showed minimal systemic spread and no mortality, as opposed to 100% death of the mice challenged with the isogenic wild-type strain. Ty2ΔRelAΔSpoT had markedly elongated morphology with incomplete septa formation and demonstrated severely attenuated motility and chemotaxis due to the loss of flagella. Absence of the Vi-polysaccharide capsule rendered the mutant strain highly susceptible to complement-mediated lysis. The phenotypes of Ty2ΔRelAΔSpoT was contributed by transcriptional repression of several genes, including fliC, tviA, and ftsZ, as found by reverse transcriptase quantitative polymerase chain reaction and gene complementation studies. Finally, Ty2ΔRelAΔSpoT had markedly reduced invasion into intestinal epithelial cells and significantly attenuated survival within macrophages. To the best of our knowledge, this was the first study that addressed SR in S. Typhi and showed that (p)ppGpp was essential for optimal pathogenic fitness of the organism.

ITPase deficiency causes a Martsolf-like syndrome with a lethal infantile dilated cardiomyopathy.

Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated "mutation negative" probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA-and by implication rI production-correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.

Binary Function of ARL3-GTP Revealed by Gene Knockouts.

UNC119 and PDEδ are lipid-binding proteins and are thought to form diffusible complexes with transducin-α and prenylated OS proteins, respectively, to mediate their trafficking to photoreceptor outer segments. Here, we investigate mechanisms of trafficking which are controlled by Arf-like protein 3 (Arl3), a small GTPase. The activity of ARL3 is regulated by a GEF (ARL13b) and a GAP (RP2). In a mouse germline knockout of RP2, ARL3-GTP is abundant as its intrinsic GTPase activity is extremely low. High levels of ARL3-GTP impair binding and trafficking of cargo to the outer segment. Germline knockout of ARL3 is embryonically lethal generating a syndromic ciliopathy-like phenotype. Retina- and rod-specific knockout of ARL3 allow to determine the precise mechanisms leading to photoreceptor degeneration. The knockouts reveal binary functions of ARL3-GTP as a key molecule in late-stage photoreceptor ciliogenesis and cargo displacement factor.

Inhibition of arterial medial calcification and bone mineralization by extracellular nucleotides: The same functional effect mediated by different cellular mechanisms.

Arterial medial calcification (AMC) is thought to share some outward similarities to skeletal mineralization and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. ATP and UTP have previously been shown to inhibit bone mineralization. This investigation compared the effects of extracellular nucleotides on calcification in VSMCs with those seen in osteoblasts. ATP, UTP and the ubiquitous mineralization inhibitor, pyrophosphate (PPi ), dose dependently inhibited VSMC calcification by ≤85%. Culture of VSMCs in calcifying conditions was associated with an increase in apoptosis; treatment with ATP, UTP, and PPi reduced apoptosis to levels seen in non-calcifying cells. Extracellular nucleotides had no effect on osteoblast viability. Basal alkaline phosphatase (TNAP) activity was over 100-fold higher in osteoblasts than VSMCs. ATP and UTP reduced osteoblast TNAP activity (≤50%) but stimulated VSMC TNAP activity (≤88%). The effects of extracellular nucleotides on VSMC calcification, cell viability and TNAP activity were unchanged by deletion or inhibition of the P2Y2 receptor. Conversely, the actions of ATP/UTP on bone mineralization and TNAP activity were attenuated in osteoblasts lacking the P2Y2 receptor. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) hydrolyses ATP and UTP to produce PPi . In both VSMCs and osteoblasts, deletion of NPP1 blunted the inhibitory effects of extracellular nucleotides suggesting involvement of P2 receptor independent pathways. Our results show that although the overall functional effect of extracellular nucleotides on AMC and bone mineralization is similar there are clear differences in the cellular mechanisms mediating these actions.

Oral administration of pyrophosphate inhibits connective tissue calcification.

Various disorders including pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI), which are caused by inactivating mutations in ABCC6 and ENPP1, respectively, present with extensive tissue calcification due to reduced plasma pyrophosphate (PPi). However, it has always been assumed that the bioavailability of orally administered PPi is negligible. Here, we demonstrate increased PPi concentration in the circulation of humans after oral PPi administration. Furthermore, in mouse models of PXE and GACI, oral PPi provided via drinking water attenuated their ectopic calcification phenotype. Noticeably, provision of drinking water with 0.3 mM PPi to mice heterozygous for inactivating mutations in Enpp1 during pregnancy robustly inhibited ectopic calcification in their Enpp1-/- offspring. Our work shows that orally administered PPi is readily absorbed in humans and mice and inhibits connective tissue calcification in mouse models of PXE and GACI PPi, which is recognized as safe by the FDA, therefore not only has great potential as an effective and extremely low-cost treatment for these currently intractable genetic disorders, but also in other conditions involving connective tissue calcification.

RNAi mediated gene silencing of ITPA using a targeted nanocarrier: Apoptosis induction in SKBR3 cancer cells.

A pure nucleotide pool is required for high-fidelity DNA replication and prevention of carcinogenesis in living cells. Human inosine triphosphatase (ITPase), encoded by the ITPA gene, plays a critical role in maintaining the purity of the cellular nucleotide pool by excluding nucleotides that enhance mutagenesis. ITPase is a nucleoside triphosphate pyrophosphatase that hydrolyzes the non-canonical nucleotides inosine triphosphate (ITP) and xanthine triphosphate (XTP). The monophosphate products of ITPase reactions are subsequently excluded from the nucleotide pool and the improper substitution of ITP and XTP into DNA and RNA is prevented. Previous studies show that deficiency in ITPA can suppress cellular growth and enhance DNA instability. In this study, we evaluated the influence of effective ITPA down-regulation on the induction of apoptosis in a human cancer cell line using folate-single wall nanotubes (SWNT) as a targeted nanocarrier. We assessed whether SWNT enhances IPTA-siRNA transfection efficiency in cancer cells using folate as a homing device. Since folate receptor is considerably overexpressed in cancer cells, conjugation of SWNTs to folate could enhance their cancer-specific penetrance. We found that nanocarrier mediated ITPA-siRNA transfection into SKBR3 cells caused significant reduction of ITPA mRNA expression level and complete down-regulation of the ITPase protein product. The silencing of ITPA led to promotion of apoptosis in SWNT-treated SKBR3 cancer cells.

ITPA deficiency and ribavirin level are still predictive of anaemia in HCV-HIV-coinfected patients receiving ribavirin combined with a first-generation DAA (ANRS HC27 study).

BACKGROUND: We aimed to determine the impact of inosine triphosphatase (ITPA) deficiency on ribavirin (RBV)-induced anaemia in HIV-HCV-coinfected patients receiving a triple therapy including the haematotoxic direct-acting antiviral agent boceprevir (BOC). METHODS: Patients of the ANRS HC27 BocepreVIH study were genotyped for two ITPA single nucleotide polymorphisms involved in ITPA deficiency. RBV trough concentration (Ctrough) was determined at week (W)4 and W8. Impact of ITPA deficiency on anaemia, RBV Ctrough, response and haematotoxicity (grade 3/4 anaemia, erythropoietin [EPO] use, RBV dose reduction or transfusion between day [D]0 and W8) was evaluated. Impact of RBV Ctrough on anaemia was also studied. RESULTS: Among the 63 genotyped patients, 33% had a predicted ITPA deficiency. ITPA deficiency was associated with a lower haemoglobin (Hb) decline both at W4 (-1.0 g/dl versus -2.1 g/dl; P=0.02) and W8 (-2.7 g/dl versus -4.1 g/dl; P=0.05). None of the patients with ITPA deficiency received EPO between D0-W8 versus 26% of patients without ITPA deficiency (P=0.01). RBV Ctrough was associated with Hb decrease both at W4 and W8 and an RBV Ctrough cutoff value of 2 µg/ml was significantly associated with a W4 Hb decline >2 g/dl. Haematotoxicity was significantly associated with a lower W4 Hb level (P=0.017), absence of ITPA deficiency (P=0.018) and higher RBV Ctrough (P=0.012). ITPA deficiency, W4 RBV Ctrough and gender were independent predictors of anaemia at W4. ITPA deficiency was not associated with virological response. CONCLUSIONS: ITPA deficiency and RBV Ctrough are still predictive of RBV-induced anaemia in HIV-HCV-coinfected patients treated with RBV combined with a first-generation direct antiviral agent.

A disease spectrum for ITPA variation: advances in biochemical and clinical research.

Human ITPase (encoded by the ITPA gene) is a protective enzyme which acts to exclude noncanonical (deoxy)nucleoside triphosphates ((d)NTPs) such as (deoxy)inosine 5'-triphosphate ((d)ITP), from (d)NTP pools. Until the last few years, the importance of ITPase in human health and disease has been enigmatic. In 2009, an article was published demonstrating that ITPase deficiency in mice is lethal. All homozygous null offspring died before weaning as a result of cardiomyopathy due to a defect in the maintenance of quality ATP pools. More recently, a whole exome sequencing project revealed that very rare, severe human ITPA mutation results in early infantile encephalopathy and death. It has been estimated that nearly one third of the human population has an ITPA status which is associated with decreased ITPase activity. ITPA status has been linked to altered outcomes for patients undergoing thiopurine or ribavirin therapy. Thiopurine therapy can be toxic for patients with ITPA polymorphism, however, ITPA polymorphism is associated with improved outcomes for patients undergoing ribavirin treatment. ITPA polymorphism has also been linked to early-onset tuberculosis susceptibility. These data suggest a spectrum of ITPA-related disease exists in human populations. Potentially, ITPA status may affect a large number of patient outcomes, suggesting that modulation of ITPase activity is an important emerging avenue for reducing the number of negative outcomes for ITPA-related disease. Recent biochemical studies have aimed to provide rationale for clinical observations, better understand substrate selectivity and provide a platform for modulation of ITPase activity.

Relationship between ITPA polymorphisms and hemolytic anemia in HCV-infected patients after ribavirin-based therapy: a meta-analysis.

BACKGROUND: There is growing evidence that variations in the gene encoding inosine triphosphate pyrophosphohydrolase (ITPase), known as inosine triphosphatase (ITPA), are related to hemolytic anemia, which is frequently observed among hepatitis C virus (HCV)-infected patients receiving ribavirin (RBV)-based therapy. We performed a meta-analysis of all eligible studies assessing ITPA gene polymorphisms related to RBV-induced hemolytic anemia in HCV-infected patients published in PubMed, Embase and the Cochrane library prior to the end of 2014. METHODS: Three outcomes were evaluated: (1) hemoglobin decline, (2) severe anemia, and (3) RBV dose reduction or treatment discontinuation. Pooled odds ratio (OR) and 95 % confidence interval (95 % CI) were estimated by either fixed or random effects models. RESULTS: Twenty-nine studies were selected from the literature search: 20 references involving 6533 individuals for hemoglobin decline, 13 references on 3764 patients for severe anemia, and 16 references on 3918 patients for RBV dose reduction or discontinuation. Significant associations with hemoglobin decline were found for rs1127354 CC [OR = 12.84 (95 % CI 7.44; 22.17)], rs7270101 AA [OR = 3.41 (95 % CI 2.08; 5.59)] and rs6051702 AA [OR = 4.43 (95 % CI 2.80; 7.00)] genotypes. Moreover, significant associations with hemoglobin decline were also found for absent [OR = 6.01 (95 % CI 4.84; 7.46)] and mild [OR = 4.68 (95 % CI 2.83; 7.74)] ITPase deficiency haplotypes. The ITPA rs1127354 CC genotype and absent ITPase deficiency haplotype were also associated with severe anemia {[OR = 7.77 (95 % CI 5.03; 12.00)] and [OR = 4.79 (95 % CI 1.69; 13.56)], respectively}. Additionally, the rs1127354 CC genotype showed significant association with RBV dose reduction or stopping treatment (OR = 2.24; 95 % CI 1.79; 2.81). CONCLUSIONS: ITPA polymorphisms increase the likelihood of developing hemolytic anemia for HCV-infected patients on RBV-based therapy, particularly rs1127354 CC and rs7270101 AA genotypes, suggesting the utility of screening for ITPA polymorphisms to avoid hematological toxicity and increase adherence to RBV-based therapy.

Long-term rescue of cone photoreceptor degeneration in retinitis pigmentosa 2 (RP2)-knockout mice by gene replacement therapy.

Retinal neurodegenerative diseases are especially attractive targets for gene replacement therapy, which appears to be clinically effective for several monogenic diseases. X-linked forms of retinitis pigmentosa (XLRP) are relatively severe blinding disorders, resulting from progressive photoreceptor dysfunction primarily caused by mutations in RPGR or RP2 gene. With a goal to develop gene therapy for the XLRP-RP2 disease, we first performed detailed characterization of the Rp2-knockout (Rp2-KO) mice and observed early-onset cone dysfunction, which was followed by progressive cone degeneration, mimicking cone vision impairment in XLRP patients. The mice also exhibited distinct and significantly delayed falling phase of photopic b-wave of electroretinogram (ERG). Concurrently, we generated a self-complementary adeno-associated viral (AAV) vector carrying human RP2-coding sequence and demonstrated its ability to mediate stable RP2 protein expression in mouse photoreceptors. A long-term efficacy study was then conducted in Rp2-KO mice following AAV-RP2 vector administration. Preservation of cone function was achieved with a wide dose range over 18-month duration, as evidenced by photopic ERG and optomotor tests. The slower b-wave kinetics was also completely restored. Morphologically, the treatment preserved cone viability, corrected mis-trafficking of M-cone opsin and restored cone PDE6 expression. The therapeutic effect was achieved even in mice that received treatment at an advanced disease stage. The highest AAV-RP2 dose group demonstrated retinal toxicity, highlighting the importance of careful vector dosing in designing future human trials. The wide range of effective dose, a broad treatment window and long-lasting therapeutic effects should make the RP2 gene therapy attractive for clinical development.

Acidosis is a key regulator of osteoblast ecto-nucleotidase pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity.

Previous work has shown that acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of phosphate (Pi ) to pyrophosphate (PPi ) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both Pi and PPi , a potent inhibitor of mineralisation, are generated from extracellular nucleotides by the actions of ecto-nucleotidases. This study investigated the expression and activity of ecto-nucleotidases by osteoblasts under normal and acid conditions. We found that osteoblasts express mRNA for a number of ecto-nucleotidases including NTPdase 1-6 (ecto-nucleoside triphosphate diphosphohydrolase) and NPP1-3 (ecto-nucleotide pyrophosphatase/phosphodiesterase). The rank order of mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 > alkaline phosphatase > ecto-5-nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. Acidosis (pH 6.9) upregulated NPP1 mRNA (2.8-fold) and protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto-nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in acid conditions (P < 0.001). Release of ATP, one of the key substrates for NPP1, from osteoblasts, was unaffected by acidosis. Further studies showed that mineralised bone formation by osteoblasts cultured from NPP1 knockout mice was increased compared with wildtypes (2.5-fold, P < 0.001) and was partially resistant to the inhibitory effect of acidosis. These results indicate that increased NPP1 expression and activity might contribute to the decreased mineralisation observed when osteoblasts are exposed to acid conditions.